Search Results (15)

Virus filtration is an essential unit operation used to validate clearance of adventitious virus during the manufacture of biopharmaceutical products such as monoclonal antibodies. Obtaining at least a 10,000-fold reduction in virus particles in the permeate is challenging as monoclonal antibodies are about half the size of the virus particles. Minute virus of mice, FDA-recommended model adventitious virus, was labeled with a fluorescent dye. Laser scanning confocal microscopy was used to determine the location of virus entrapment within the virus filtration membrane. Three different hollow fiber membranes made of regenerated cellulose and polyvinylidene fluoride were tested. Feed streams consisted of MVM spiked in buffer and MVM spiked in 5 g L⁻¹ bovine serum albumin known to contain aggregates similar in size to the MVM. After filtering the feed, a buffer flush was used, with and without 30 min pause before the buffer flush. For all virus filters, a 30 min process pause led to broadening and movement of the virus entrapment zone deeper into the membrane. The presence of aggregates led to greater broadening of the entrapment zone. Both effects could lead to reduced virus clearance. Visualization of virus entrapment helps improve understanding of the behavior of virus filtration membranes. Full article

Datong in Shanxi Province, known as the “Daylily Capital of China,” still primarily relies on traditional propagation by division for daylily seedling production. Although traditional seedling propagation methods are simple and low-cost, they suffer from limitations such as low propagation efficiency, which restricts large-scale production. The application of tissue culture in seedling production not only enables rapid large-scale propagation but also helps maintain desirable genetic traits through virus elimination. This study aimed to establish an efficient in vitro regeneration system for Hemerocallis citrina ‘Datong Huanghua’ through optimization of key culture stages using scape explants. The results demonstrated that during the stages of callus induction, adventitious bud differentiation, and proliferation culture, the best results were achieved using MS medium supplemented with 3 mg/L zeatin (ZT) and 0.3 mg/L α-naphthylacetic acid (NAA), yielding a callus induction rate of 83.33%, an adventitious bud differentiation rate of 83.40%, and a proliferation coefficient of 4.05. For root induction, MS medium containing 0.25 mg/L indole-3-butyric acid (IBA) and 0.25 mg/L NAA resulted in an average of 4.7 roots per plantlet with a 100% rooting rate. In addition, endogenous hormone analysis showed that lower ABA/GA₃ and ABA/ZR ratios in scape explants promoted callus formation during the induction and differentiation stages. Full article

Vegetative propagation through stem cuttings represents the primary mode of reproduction in Rosa species. While numerous studies have reported physiological factors affecting cutting rooting, the genes regulating the formation of adventitious roots in roses have not yet been fully explored and studied. In this study, we demonstrate that Rosa rugosa ‘Feng Hua’ exhibits an indirect rooting pattern, requiring callus formation prior to root primordium differentiation. Phytohormone profiling revealed exceptionally high concentrations of auxin precursors, particularly tryptophan (Trp), in both callus and root tissues. Therefore, we identified and analyzed the members of the YUCCA family, which are the key rate-limiting enzymes in the tryptophan-dependent IAA biosynthesis pathway. A total of 11 RrYUCs family genes were identified, with RT-qPCR analysis showing that RrYUC10 was highly expressed in callus and root tissues. Functional studies confirmed its critical role in adventitious root formation: virus-induced gene silencing (VIGS) of RrYUC10 significantly inhibited AR development, whereas its overexpression enhanced rooting. Our findings have provided a molecular theoretical basis for the rooting of cuttings in roses. Full article

Witches’ broom disease of blue palo verde (Parkinsonia florida) was reported more than sixty years ago. Characteristic symptoms consist of dense clusters of shortened, brittle branches and stunted leaves. The suspect causal agent has been identified as palo verde broom virus (PVBV), genus, Emaravirus, family, Fimoviridae. Here, the first complete PVBV genome sequence was determined, and virus small interfering RNAs (vsiRNAs), primary metabolites, and phytohormone profiles were characterized from infected palo verde leaves, adventitious shoots, flowers, and seeds. Based on pairwise distances, PVBV RNAs 1–4 shared 54–65% nucleotide identity and 19–51% amino acid similarity, respectively, with other emaraviruses, while PVBV RNA 5 shared no sequence homology with any emaravirus. The 21–24-nt virus-derived vsiRNAs, indicative of post-transcriptional gene silencing (PTGS), represented nearly the entire PVBV genome in flowers, leaves, seeds, and adventitious shoots; however, PVBV RNA 3 and RNA 4 were most heavily targeted in all plant parts. Evidence that six major phytohormones were altered in PVBV-infected compared to virus-free trees indicated that emaravirus-infected trees mount classical defense responses to virus infection and/or eriophyid mite infestations. Detection of PVBV RNA genome segments 1–5, accumulation of predominantly 21-nt vsiRNAs, homologous to the PVBV genome and transcripts, and altered levels of phytohormones and metabolites in PVBV-infected trees strongly implicate PVBV as the causal agent of witches’ broom disease. Full article

Taro (Colocasia esculenta (L.) Schott) is the fifth largest rhizome crop, and it is widely distributed in tropical and subtropical areas in the world. Vegetative propagation with virus-infected corms can lead to cultivar degradation, yield decline, and quality deterioration. In this study, the shoot apical meristems excised from taro corms infected with dasheen mosaic virus, which belongs to the genus Potyvirus in the family Potyviridae, were cultured and treated with exogenous abscisic acid and high sucrose concentrations to induce micro-corm formation. Subsequently, candidate genes involved in micro-corm expansion were screened via transcriptome sequencing analysis. The results revealed that the shoot apical meristems could grow into adventitious shoots on the medium 1 mg/L 6-benzylaminopurine + 0.3 mg/L 1-naphthaleneacetic acid, and reverse transcription–polymerase chain reaction detection indicated that dasheen mosaic virus had been successfully eliminated from the test-tube plantlets. Moreover, 8% sucrose or 3% sucrose + 5 μM abscisic acid likewise induced taro corm formation, and genes related to cell division and the cell cycle, as well as starch and sucrose metabolism pathways, were significantly enriched during taro corm expansion. Furthermore, the cyclin-dependent kinases genes, cell cycle protein kinase subunit genes, and cyclin B2 genes, which are related to cell division and the cell cycle, were upregulated with abscisic acid treatment on the 3rd day. The sucrose synthase genes, β-amylase genes, glycogen branching enzyme genes, and soluble starch synthase genes, which are related to starch and sucrose metabolism, were upregulated on the 15th day, indicating that cell division largely occurs during taro corm formation, whereas carbohydrates are synthesized during taro corm expansion. Full article

The coronavirus disease-19 (COVID-19) pandemic, declared in early 2020, has left an indelible mark on global health, with over 7.0 million deaths and persistent challenges. While the pharmaceutical industry raced to develop vaccines, the emergence of mutant severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) strains continues to pose a significant threat. Beyond the immediate concerns, the long-term health repercussions of COVID-19 survivors are garnering attention, particularly due to documented cases of cardiovascular issues, liver dysfunction, pulmonary complications, kidney impairments, and notable neurocognitive deficits. Recent studies have delved into the pathophysiological changes in various organs following post-acute infection with murine hepatitis virus-1 (MHV-1), a coronavirus, in mice. One aspect that stands out is the impact on the skin, a previously underexplored facet of long-term COVID-19 effects. The research reveals significant cutaneous findings during both the acute and long-term phases post-MHV-1 infection, mirroring certain alterations observed in humans post-SARS-CoV-2 infection. In the acute stages, mice exhibited destruction of the epidermal layer, increased hair follicles, extensive collagen deposition in the dermal layer, and hyperplasticity of sebaceous glands. Moreover, the thinning of the panniculus carnosus and adventitial layer was noted, consistent with human studies. A long-term investigation revealed the absence of hair follicles, destruction of adipose tissues, and further damage to the epidermal layer. Remarkably, treatment with a synthetic peptide, SPIKENET (SPK), designed to prevent Spike glycoprotein-1 binding with host receptors and elicit a potent anti-inflammatory response, showed protection against MHV-1 infection. Precisely, SPK treatment restored hair follicle loss in MHV-1 infection, re-architected the epidermal and dermal layers, and successfully overhauled fatty tissue destruction. These promising findings underscore the potential of SPK as a therapeutic intervention to prevent long-term skin alterations initiated by SARS-CoV-2, providing a glimmer of hope in the battle against the lingering effects of the pandemic. Full article

The transcriptional properties of artificial promoters are closely related to the type and arrangement position of cis-elements. GWSF (374-bp) was an effective SPIP with four cis-element dimers. There were four pathogen-inducible cis-elements in the GWSF promoter (GST1-boxes, W-boxes, S-boxes, and F-boxes) and a minimal cauliflower mosaic virus 35S promoter. V-element dimers were inserted into the upstream (VGWSF), midstream (GWVSF), and downstream (GWSFV) regions of the original GWSF promoter sequence to examine their affect on the position. The expression activity of promoters was analyzed and estimated using the histochemical staining of leaf discs of eucalyptus with transient expression, an image digitization method to extract the color features, and the induction treatment by a plant pathogenic microorganism/inducer and qPCR assays. The histochemical staining results of the adventitious buds indicated that the promoters had been successfully integrated into the E. urophylla genome and that they drove the expression of the gus gene. There was a noticeable difference in the intensity of color between the adventitious buds on the same callus block, as well as the intensity of color within the same adventitious bud. According to the established two-factor model of blue value, there was a greater difference between the levels of the genotype factor than the promoter factor in eucalyptus leaf discs. Further, the basal and inducible transcriptional levels of the three improved promoters were investigated by qPCR. With the basal transcriptional level of the GWSF promoter normalized to one, the relative basal levels of VGWSF, GWVSF, and GWSFV were 1.40, 1.45, and 4.15, respectively. The qPCR results were consistent with the staining results of GUS histochemical staining. The three improved promoters all had the properties of being induced by salicylic acid, Ralstonia solanacearum, and Phytophthora capsici. The three improved promoters demonstrated a significantly higher TMV induction activity: their induction activity from high to low was GWSFV > GWVSF > VGWSF. The findings will be beneficial to the construction and optimization of artificial promoters for transgenic plants. Full article

Vaccinations to prevent infectious diseases are given to target the body’s innate and adaptive immune systems. In most cases, the potency of a live virus vaccine (LVV) is the most critical measurement of efficacy, though in some cases the quantity of surface antigen on the virus is an equally critical quality attribute. Existing methods to measure the potency of viruses include plaque and TCID50 assays, both of which have very long lead times and cannot provide real time information on the quality of the vaccine during large-scale manufacturing. Here, we report the evaluation of LumaCyte’s Radiance Laser Force Cytology platform as a new way to measure the potency of LVVs in upstream biomanufacturing process in real time and compare this to traditional TCID50 potency. We also assess this new platform as a way to detect adventitious agents, which is a regulatory expectation for the release of commercial vaccines. In both applications, we report the ability to obtain expedited and relevant potency information with strong correlation to release potency methods. Together, our data propose the application of Laser Force Cytology as a valuable process analytical technology (PAT) for the timely measurement of critical quality attributes of LVVs. Full article

In the presence of temporal arteritis, clinicians often refer to the diagnosis of giant cell arteritis (GCA). However, differential diagnoses should also be evoked because other types of vascular diseases, vasculitis or not, may affect the temporal artery. Among vasculitis, Anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis is probably the most common, and typically affects the peri-adventitial small vessel of the temporal artery and sometimes mimics giant cell arteritis, however, other symptoms are frequently associated and more specific of ANCA-associated vasculitis prompt a search for ANCA. The Immunoglobulin G4-related disease (IgG4-RD) can cause temporal arteritis as well. Some infections can also affect the temporal artery, primarily an infection caused by the varicella-zoster virus (VZV), which has an arterial tropism that may play a role in triggering giant cell arteritis. Drugs, mainly checkpoint inhibitors that are used to treat cancer, can also trigger giant cell arteritis. Furthermore, the temporal artery can be affected by diseases other than vasculitis such as atherosclerosis, calcyphilaxis, aneurysm, or arteriovenous fistula. In this review, these different diseases affecting the temporal artery are described. Full article

High-throughput sequencing (HTS) allows detection of known and unknown viruses in samples of broad origin. This makes HTS a perfect technology to determine whether or not the biological products, such as vaccines are free from the adventitious agents, which could support or replace extensive testing using various in vitro and in vivo assays. Due to bioinformatics complexities, there is a need for standardized and reliable methods to manage HTS generated data in this field. Thus, we developed LABRADOR—an analysis pipeline for adventitious virus detection. The pipeline consists of several third-party programs and is divided into two major parts: (i) direct reads classification based on the comparison of characteristic profiles between reads and sequences deposited in the database supported with alignment of to the best matching reference sequence and (ii) de novo assembly of contigs and their classification on nucleotide and amino acid levels. To meet the requirements published in guidelines for biologicals’ safety we generated a custom nucleotide database with viral sequences. We tested our pipeline on publicly available HTS datasets and showed that LABRADOR can reliably detect viruses in mixtures of model viruses, vaccines and clinical samples. Full article

The induction of adventitious organs, such as calli, shoots, and somatic embryos, in tissue culture is a useful technique for plant propagation and genetic modification. In recent years, several genes have been reported to be adventitious organ inducers and proposed to be useful for industrial applications. Even though the Arabidopsis (Arabidopsis thaliana) WUSCHEL (WUS) and LEAFY COTYLEDON 1 (LEC1) genes can induce adventitious organ formation in Arabidopsis without phytohormone treatment, further improvement is desired. Here, we show that modifying the transcriptional repression/activation activities of WUS and LEC1 improves the efficiency of adventitious organ formation in Arabidopsis. Because WUS functions as a transcriptional repressor during the induction of adventitious organs, we fused it to an artificial strong repression domain, SUPERMAN REPRESSION DOMAIN X (SRDX). Conversely, we fused the strong transcriptional activation domain VP16 from herpes simplex virus to LEC1. Upon overexpression of the corresponding transgenes, we succeeded in improving the efficiency of adventitious organ induction. Our results show that the modification of transcriptional repression/activation activity offers an effective method to improve the efficiency of adventitious organ formation in plants. Full article

Prunus spp. is one of the most recalcitrant fruit tree species in terms of in vitro regeneration and transformation, mostly when mature tissues are used as explants. The present study describes the in vitro regeneration via indirect organogenesis, and Agrobacterium tumefaciens-mediated transformation of the peach rootstock Hansen 536 (Prunus persica × Prunus amygdalus) through the use of meristematic bulks (MBs) as starting explants. Efficient adventitious shoot regeneration was obtained when Hansen 536 MBs were cultured on an optimized medium consisting of modified McCown Woody Plant medium (WPM) enriched with 4.4 μM 6-Benzyladenine (BA), 0.1 μM 1-Naphthaleneacetic acid (NAA) and 6.0 g L⁻¹ plant agar S1000 (B&V). MB slices were used later as starting explants for Agrobacterium-mediated transformation to introduce an RNAi construct “ihp35S-PPV194” against PPV virus. Transgenic events were identified by both green fluorescent protein (GFP) screening and kanamycin selection at different concentrations (0, 17 or 42 μM). GFP-fluorescent proliferating callus lines were selected and confirmed to stably express the ihp35S-PPV194::eGFP gene construct by molecular analysis. Although shoot regeneration from these transgenic calli has not been obtained yet, this represents one of the few examples of successful attempts in peach genetic transformation from somatic tissues, and also serves as a useful in vitro system for future gene functional analysis in peach. Full article

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation. Full article

High-throughput sequencing (HTS) has demonstrated capabilities for broad virus detection based upon discovery of known and novel viruses in a variety of samples, including clinical, environmental, and biological. An important goal for HTS applications in biologics is to establish parameter settings that can afford adequate sensitivity at an acceptable computational cost (computation time, computer memory, storage, expense or/and efficiency), at critical steps in the bioinformatics pipeline, including initial data quality assessment, trimming/cleaning, and assembly (to reduce data volume and increase likelihood of appropriate sequence identification). Additionally, the quality and reliability of the results depend on the availability of a complete and curated viral database for obtaining accurate results; selection of sequence alignment programs and their configuration, that retains specificity for broad virus detection with reduced false-positive signals; removal of host sequences without loss of endogenous viral sequences of interest; and use of a meaningful reporting format, which can retain critical information of the analysis for presentation of readily interpretable data and actionable results. Furthermore, after alignment, both automated and manual evaluation may be needed to verify the results and help assign a potential risk level to residual, unmapped reads. We hope that the collective considerations discussed in this paper aid toward optimization of data analysis pipelines for virus detection by HTS. Full article

Cytomegalovirus (CMV) is a prevalent pathogen in the immunocompromised host and invasive pneumonia is a feared complication of the virus in this population. In this pediatric case series we characterized CMV lung infection in 15 non-HIV infected children (median age 3 years; IQR 0.2–4.9 years), using current molecular and imaging diagnostic modalities, in combination with respiratory signs and symptoms. The most prominent clinical and laboratory findings included cough (100%), hypoxemia (100%), diffuse adventitious breath sounds (100%) and increased respiratory effort (93%). All patients had abnormal lung images characterized by ground glass opacity/consolidation in 80% of cases. CMV was detected in the lung either by CMV PCR in bronchoalveolar lavage (82% detection rate) or histology/immunohistochemistry in lung biopsy (100% detection rate). CMV caused respiratory failure in 47% of children infected and the overall mortality rate was 13.3%. Conclusion: CMV pneumonia is a potential lethal disease in non-HIV infected children that requires a high-index of suspicion. Common clinical and radiological patterns such as hypoxemia, diffuse adventitious lung sounds and ground-glass pulmonary opacities may allow early identification of CMV lung infection in the pediatric population, which may lead to prompt initiation of antiviral therapy and better clinical outcomes. Full article